BACKGROUND: Mechanical ventilation (MV) is used to provide respiratory support to >15 million patients annually during both critical illnesses and surgery. Although MV is often a life-saving intervention, prolonged MV (?12 hours) promotes problems in ?weaning? patients from the ventilator; this failure to wean significantly increases patient morbidity and mortality. While the cause of difficult weaning can be multifactorial, weak inspiratory muscles (i.e., diaphragm) are a major factor. Indeed, an unintended consequence of ventilator support is that prolonged MV results in diaphragmatic inactivity and the rapid development of diaphragm atrophy and contractile dysfunction (collectively known as ventilator-induced diaphragm dysfunction (VIDD)). VIDD occurs due to both decreased protein synthesis and increased protein breakdown in the diaphragm; however, the upstream regulators that control these events remain unclear. Our compelling preliminary data suggest that the cysteine protease calpain plays an essential signaling role in the development of VIDD. Indeed, our pilot experiments reveal that prevention of calpain activation in the diaphragm protects against VIDD, indicating that calpain is required for the development of VIDD. Importantly, these results challenge the current dogma that the role of the classical calpains (i.e., calpain 1 and 2) in skeletal muscle atrophy is limited to cleavage of cytoskeletal proteins. The mechanisms by which active calpain promotes VIDD remain unknown and are the focus of this R21 application. HYPOTHESIS: Guided by our preliminary experiments, we hypothesize that active calpain is an essential upstream regulator of both protein synthesis and proteolysis in diaphragm muscle during prolonged MV. SPECIFIC AIMS: Aim 1 will determine if active calpain plays an essential role in MV-induced decreases in anabolic signaling (i.e., Akt/mTOR) and protein synthesis in the diaphragm during prolonged MV. Aim 2 will establish whether active calpain regulates MV-induced proteolysis in the diaphragm via activation of caspase-3 and forkhead box O (FoxO) transcription factors. APPROACH: We will test our hypothesis by utilizing a well- established animal model of MV along with innovative molecular tools to prevent MV-induced calpain activation in the diaphragm. Cause and effect will be determined using an adeno-associated virus vector (AAV9) to overexpress a calpastatin transgene in the diaphragm that will prevent MV-induced activation of calpain. Primary dependent measures include key steps in anabolic and proteolytic signaling, in vivo protein synthesis, rate of diaphragm proteolysis, and assessment of both caspase-3 activity along with the transcriptional activity of FoxO in the diaphragm. RELEVANCE: VIDD is a serious clinical problem because inspiratory muscle weakness is a major cause of the inability to wean patients from the ventilator. Unfortunately, no standard clinical therapy exists to prevent VIDD. These exciting exploratory experiments will provide novel and important information regarding the role that calpain signaling plays in VIDD. Successful completion of these important experiments will lead to new therapeutic strategies in the prevention of VIDD.